Chronic gastric injury leads to alterations in the differentiation of key cell lineages that predispose to more severe disease (e.g., cancer). Chronically injured gastric epithelium is characterized by cell lineage drop-out (atrophy) and aberrant differentiation (metaplasia). There is growing evidence that the digestive-enzyme secreting zymogenic cell (ZC) is lost in gastric atrophy, and furthermore, that alterations in the ZC lineage play a role in metaplasia. However, little is known about the cellular or molecular processes regulating ZC differentiation either in normal gastric units or in chronic injury. Our long-term objective is to understand ZC differentiation at the molecular and cellular level in hopes of better understanding how gastric epithelial differentiation goes awry in chronic injury. Our recent work has shown the first transcription factor, Mist1, which plays a role in ZC differentiation. Analysis of Mist1 null mice indicates that, in the absence of Mist1, ZCs do not mature normally. Rather, Mist1-/- ZCs have remarkably stunted apical cytoplasms characterized by apical plasma membrane projections and accumulation of cytoskeletal proteins like myosin and the general cytoskeletal adaptor protein Cd2ap (CD2-associated protein). We also show that the cells in the bases of a mouse model of atrophy show metaplasia, characterized by partial, aberrant ZC differentiation. Interestingly, these metaplastic cells lack Mist1 and have increased Cd2ap. The goals of the current grant are focused on determining the molecular and cellular mechanisms of Mist1 action in normal gastric units, in a model of atrophy, and in vitro in human gastric cell lines. The aims of the grant are to: 1) to determine how Mist1 interacts with Cd2ap at the molecular level (specifically, is Cd2ap transcriptionally regulated by Mist1?) in vitro in human gastric cancer cells and in vivo in Cd2ap/Mist1 double knockout mice; 2) to determine whether Mist1 expression is sufficient to drive cytoskeletal and/or proliferative changes in cells; and 3) to identify new targets of Mist1 by laser-capture microdissecting Mist1-/- and wildtype ZC lineage cells followed by microarray identification of genes with Mist1-dependent expression changes. We propose that Mist1 is the bellwether for gastric injury, i.e., that loss of its expression is a first hit that indicates the gastric unit is on a path to aberrant differentiation. Thus, if we understand how Mist1 works, especially what genes it interacts with, we might be able eventually to treat or prevent metaplasia or atrophy. Little is known about the molecules and genes that predispose people to developing gastric cancer. This project aims to further our understanding of the genes that maintain normal renewal of the digestive enzyme secreting cells in the stomach lining, because it is these cells that seemingly are the first to become aberrant on the way to tumor development.